Peptides derived from human bplp protein, polynucleotides coding for said peptides and antibodies directed against said peptides

ABSTRACT

The invention relates to an in vitro method for prognosis, diagnosis or determination of the evolution of a condition involving an altered production of Basic Proline-rich Lacrimal Protein (BPLP) or of any of its maturation products, by detecting, or quantifying in a biological sample of a test subject, a BPLP protein or a maturation product thereof, and comparing the production of BPLP protein or maturation product with the production of the same in a biological sample of a control subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/593,071, filed onJan. 19, 2007, which is a National Stage (371) of PCT/IB2005/000700,filed on Mar. 18, 2005, which claims priority to EP 04290754.3, filed onMar. 19, 2004.

FIELD OF THE INVENTION

The present invention relates to peptides derived from human BPLPprotein, as new inhibitors of metallo-ectopeptidases. The presentinvention also relates to polynucleotides coding for said peptides andto antibodies directed against said peptides. Furthermore, the presentinvention relates to diagnostic and therapeutic uses of human BPLPprotein, peptides derived therefrom and mimetics thereof, polypeptidescoding for human BPLP protein or peptides derived therefrom as well asantibodies directed against BPLP protein or peptides derived therefrom.

BACKGROUND OF THE INVENTION

In a genomic approach, an androgen-regulated gene, which ispredominantly expressed in the submandibular gland (SMG) and prostate ofadult rats, has been identified (Rosinski-Chupin et al., 1988 andEuropean patent 0 394 424). The gene encodes a precursor protein,submandibular rat₁ protein (SMR₁) giving rise to three structurallyrelated peptides which are selectively matured from the precursor invivo by cleavage at multibasic sites by a paired basic aminoacid-converting enzyme (Rougeot et al., 1994).

In an approach of post-genomic and physiomic, it was established themolecular and functional bases providing evidence for the existence inmammals of a hormonal messenger of the intercellular communication,i.e., the final mature peptide generated from SMR₁ pre-prohormone:SMR1-Pentapeptide, named today Sialorphin (of sequence QHNPR (SEQ ID NO:8)). Hence, sialorphin is an exocrine and endocrine peptide-signal,whose expression is under activational androgenic regulation andsecretion is evoked under adrenergic-mediated response to environmentalstress, in male rat (Rougeot et al., 1997).

The fact that, in sexually mature male rat, the androgen-regulatedsialorphin is acutely secreted in response to environmental acutestress, led to postulate that this signaling mediator might play a rolein some physiological and behavioral integration linked to thereproduction. Thus the same authors investigated the effects induced bysialorphin on the male sexual behavior pattern, which included frequencyand latency of mounts, intromissions and ejaculations, as well associo-sexual interactions. The data obtained showed that sialorphin hasthe ability to modulate, at doses related to physiological circulatinglevels, the male rat mating pattern, i.e., exerting, in a dose-dependentmanner, a dual facilitative/inhibitory effect on the sexual performance,while stimulating at all doses the apparent sexual arousal ormotivation. Thus it is proposed that the endogenous androgen-regulatedsialorphin helps modulate the adaptative balance between excitatory andinhibitory mechanisms serving appropriate male rat sexual response,depending on the context.

International patent application WO 01/00221 describes the use ofmaturation products of SMR1 for the treatment of impaired interpersonaland behavioural disorders, including sexual defects.

Furthermore, these authors discovered that SMR1 maturation productsrecognize specific target sites in organs that are deeply involved inthe mineral ion concentration. International patent application WO98/37100 describes the therapeutic use of maturation products of SMR1for preventing or treating diseases associated with a mineral ionimbalance in a human or an animal body.

In response to stressful contexts, sialorphin is acutely released,rapidly distributed and lasting taken up by its systemicmembrane-associated targets (Rougeot et al., 1997). The authors havedemonstrated that the major cell surface molecule to which sialorphinbinds in vivo is the membrane-anchored metalloecto-endopeptidase, NEP(Neutral Endopeptidase; Neprilysin EC 3.4.24.11), or enkephalinase(Rougeot et al., 2003). Moreover, sialorphin was shown to be aphysiological antagonist of the NEP activity ex vivo; and the directinteraction of NEP and sialorphin assessed in an in vitro assay usingsoluble purified renal NEP and artificial fluorogenic DGNPA(Dansyl-Gly-(pNO2)Phe-βAla) as substrate provided direct evidence thatsialorphin inhibited NEP activity (IC 50 of the sialorphin: 0.6 μM).Sialorphin, is the first physiological inhibitor of theNEP-enkephalinase activity identified to date in rodent (Rougeot et al.,2003 and European patent application EP 1 216 707).

NEP is located at the surface of cells in nervous and systemic tissues,where it plays an important function as an ectoenzyme catalyzing thepost-secretory processing or metabolism of a number of neuropeptides andregulatory peptides. The main physiologically relevant substrates forNEP are the enkephalins, substance P and atrial natriuretic peptide(ANP). These mammalian signal peptides are involved in the control ofcentral and peripheral pain perception, inflammatory phenomena, arterialtone and mineral homeostasis. Their physiological importance and thecritical role of NEP ectoenzyme in modulating their functional potencymake it important to investigate and know their possible protection byendogenous inhibitors, from a physiological as well as aphysiopathological and therapeutic point of view.

By using different models of molecular and behavioral pharmacology, theauthors have shown that the physiological mediator, sialorphin, preventsspinal and renal NEP from breaking down its two physiologically relevantsubstrates, Substance P and Met-enkephalin in vitro. Sialorphininhibited the breakdown of substance P with an IC50 of 0.4-1 μM andbehaved as a competitive inhibitor of the membrane-bound NEP thatoriginates from nervous tissues (spinal cord) or from systemicallytissues (kidney, bone, tooth, placenta, prostate, GSM, intestine). Invivo, intravenous sialorphin elicited potent antinociceptive responsesin two behavioral rat models of injury-induced acute and tonic pain, thepin-pain test (mechanical algesia) and formalin test (chemical algesia).The analgesia induced by sialorphin required the activation of μ- andδ-opioid receptors, consistent with the involvement of endogenous opioidreceptors in enkephalinergic transmission. Indeed, these receptors areinvolved in the transmission of the endogenous opioidergic signals suchas the enkephalins which are inactivated by NEP and the aminopeptidaseAPN, and also of the exogenous opiate, the morphine which interactsmainly with the μ-opioid receptor. It was concluded that the sialorphinprotects endogenous enkephalins released following nociceptive stimuliby inhibiting ecto-enkephalinases, in vivo, and thus potentialises theiranalgesic effect. Otherwise, the endogenous opioid system, in particularδ-opioid-mediated pathway, has also been linked to the etiology ofdepressive behavior; for instance using a model of analysis ofbehavioral despair (forced swim test), the authors showed thatsialorphin displays a significative antidepressant activity in male rat.Sialorphin is the first natural systemically active regulator of NEPactivity identified to date in mammals. Furthermore, evidence wasprovided that it is a new physiological modulator of pain perceptionfollowing injury, and may be the progenitor of a new class oftherapeutic molecules, as putative novel antinociceptive andantidepressive agents (Rougeot et al., 2003; EP 1,343,519 and EP1,343,520).

The powerful analgesic effect of sialorphin is associated to itscapacity to entirely protect the enkephalins from inactivation by theenkephalin-degrading ectoenzymes. In vivo, the enkephalins areinactivated with an extraordinarily efficiency (within few seconds) bythe both ectopeptidases, NEP and APN. In agreement, the first developedsynthetic inhibitors, which are either only NEP specific (such asThiorphan) or APN specific (such as Bestatin) exhibit a non-significantor weak antinociceptive effect. Thus, rat sialorphin is a physiologicaldual inhibitor of NEP and APN metallo-ectopeptidases; furthermore, thisendocrine signal messenger of the adaptative response to stress is apowerful inhibitor of painful perception in rat and its analgesic effectis more potent than that of synthetic dual NEP/APN inhibitors such askelatorphan, which have been developed elsewhere by modeling methods.So, sialorphin is remarkably adapted in terms of specificity andbioavailability to the conformational and distributive characteristicsof its targets and as a consequence is more effective from anintegrative point of view. Considering these observations, from afunctional as well as physiopathological and therapeutic point of view,the biological importance of the functions regulated by the ratsialorphin makes it crucial to investigate and identify the endogenousfunctional homologous of rat sialorphin in human.

Sialorphin is the only identified physiological systemically activeregulator of the membrane-bound enkephalinase activity in mammals. Thisraises the question of the existence of such endogenousNEP-ectopeptidase inhibitor in human saliva and blood. No immunoreactiveQHNPR peptide (SEQ ID NO: 8) (sialorphin) was detected in male humansaliva using highly sensitive and specific radioimmunoassay (Rougeot etal., 1994). However, bibliographical data let suppose the presence oflow molecular weight substances (≦3000 Da), inhibiting the NEPectopeptidase activity in human, notably in the human saliva. Althoughthis(ese) salivary component(s) was(were) not biochemicallycharacterized, a gender-related difference was observed in the salivaryproduction of this(ese) inhibitor(s) of human enkephalin-degradingectoenzymes (Marini and Roda, 2000). Strikingly, the situation is verysimilar to that one identified by the inventors in male rat, wherein thesubmandibular gland and the saliva represented the compartments of majorsynthesis and secretion of sialorphin, respectively.

The gene encoding the SMR1 precursor of sialorphin belongs to amultigene family whose members have been identified in human. However,the stricto sensu homologous human gene of rat SMR1 gene (VCSA1 codingfor SMR1) was not found in human (cDNA cloning and human genomeanalysis). Furthermore, the inhibitory potency of rat sialorphin againstmembrane-anchored human NEP, which is expressed by human prostate celllines (LNCaP), exists but is about 10-fold lower than that obtainedagainst rodent NEP (rat, rabbit). This apparent selectivity in thefunctional interaction between rat sialorphin and NEP ectoenzyme is atleast surprising considering the fact that the rat and human NEP haverelatively high amino-acid sequence analogy (about 85%). Otherwise, thecharacterization of the human genes of the multigenic family to whichbelongs the gene coding for the precursor of the rat sialorphin (SMR1),revealed that it exists in human, several genes of this family, amongwhich three were characterized, i.e., the genes hPB, hPBI and BPLP whichare clustered in the same chromosome region, q13-21 of Chromosome 4(Isemura, 2000) (Isemura and Saitoh, 1997) (Dickinson and Thiesse,1996).

DESCRIPTION OF THE INVENTION

The inventors have now identified a new peptide that is considered asthe functional human homologous of the SMR1-pentapeptide sialorphin.

The numerous data collected by the inventors support that the newpeptide, of sequence QRFSR (SEQ ID NO: 3), derives from the BPLP protein(“Basic Proline-rich Lacrimal Protein”).

The human gene BPLP codes for a polypeptide sequence of 201 amino-acids(with the potential signal peptide of secretion) predicted from the cDNAcloned and characterized by Dickinson and al. (Dickinson and Thiesse,1996). The gene BPLP is expressed in human lacrimal and submandibularglands. In the annexed sequence listing, SEQ ID No. 1 shows the cDNAsequence coding for BPLP, and SEQ ID No. 2 shows the BPLP aminoacidsequence.

The inventors defined consensus sites in the best conserved N-terminalregion (between the rat, mouse and human) of the secreted BPLP protein,on the basis of the maturation processing of rat sialorphin from theSMR1 precursor.

For instance, these consensus sites were defined as signal peptidecleavage sites in a region having the sequence required for the signalpeptidase and at paired basic residues with R—R bonds recognized asprocessing signal for paired basic amino acid-convertase.

At such consensus sites, the inventors then found out a sequence QRFSR(SEQ ID NO: 3), structurally closely related to that of rat QHNPR (SEQID NO: 8) sialorphin.

This peptide was synthesized and analyzed for its capacity to inhibitthe degradation of the physiological NEP substrate, i.e. substance P.

This peptide was then identified as the human functional homologous ofsialorphin.

The present invention is drawn to peptides derived from human BPLPprotein, as new inhibitors of metallo-ectopeptidases.

More particularly, the present invention is drawn to maturation productsof the BPLP protein, in particular the QRFSR peptide (SEQ ID NO: 3), aswell as peptide derivatives and mimetics thereof, useful to potentialisethe effects of neuroendocrine peptide messengers which control thenociceptive transmission (e.g. enkephalins), the well-being and/or thehomeostatic exchanges of Na/Pi/Ca/H₂O mainly (e.g. natriureticpeptides).

The present invention is also drawn to polynucleotides coding for saidpeptides and peptide derivatives as well as to antibodies directedagainst said peptides and peptide derivatives thereof.

Furthermore, the present invention is drawn to diagnostic andtherapeutic uses of human BPLP protein, human BPLP protein-derivedpeptides, and peptides derivatives and mimetics thereof, as well asdiagnostic and therapeutic uses of polynucleotides coding for human BPLPprotein, human BPLP protein-derived peptides and peptides derivativesthereof and of antibodies directed against human BPLP protein, humanBPLP protein-derived peptides and peptide derivative thereof.

It should be understood that the peptides, proteins, or nucleic acids ofthe invention are in isolated or purified form.

By <<purified>> and <<isolated>> it is meant, when referring to aprotein or peptide (including antibodies) or a nucleotide sequence, thatthe indicated molecule is present in the substantial absence of otherbiological molecules. The term “purified” as used herein preferablymeans at least 75% by weight, more preferably at least 85% by weight,more preferably still at least 95% by weight, and most preferably atleast 98% by weight, of biological molecules of the same type arepresent. An “isolated” or “purified” nucleic acid molecule which encodesa particular polypeptide refers to a nucleic acid molecule which issubstantially free of other nucleic acid molecules that do not encodethe subject polypeptide. However, the molecule may include someadditional bases or moieties which do not deleteriously affect the basiccharacteristics of the composition.

Peptides

For purposes of the invention, a “peptide” is a molecule comprised of alinear array of amino acid residues connected to each other in thelinear array by peptide bonds. Such linear array may optionally becyclic, i.e., the ends of the linear peptide or the side chains of aminoacids within the peptide may be joined, e.g., by a chemical bond. Suchpeptides according to the invention may include from about three toabout 500 amino acids, preferably from about 3 to about 100 amino acids,and most preferably from about 3 to about 50 amino acids and especiallyfrom about 3 to 15 amino acids and may further include secondary,tertiary or quaternary structures, as well as intermolecularassociations with other peptides or other non-peptide molecules. Suchintermolecular associations may be through, without limitation, covalentbonding (e.g., through disulfide linkages), or through chelation,electrostatic interactions, hydrophobic interactions, hydrogen bonding,ion-dipole interactions, dipole-dipole interactions, or any combinationof the above.

In these peptides, by N-terminal cyclization/decyclization, Glp and Glninterconvert.

A subject of the present invention is a peptide which is derived fromhuman BPLP protein and which has a modulatory, especially an inhibitoryactivity on metallo-ectopeptidases.

“Derived from human BPLP protein” means which comprises, consistsessentially of, or consists of a BPLP protein fragment. In a preferredembodiment, said peptide consists of 3 to about 150 amino acids. Mostpreferably, said peptide consists of less than 100 amino acids.

Particularly a subject of the present invention is a maturation productof the BPLP protein as well as peptide derivatives thereof.

More particularly, it is drawn to a peptide that is a maturation productof the Basic Proline-rich Lacrinal Protein (BPLP) or peptide derivativeof said maturation product, wherein the peptide or peptide derivativesexhibits an inhibitory property against a metallo-ectopeptidase,especially NEP and/or APN, and more particularly NEP.

A “maturation product” is a peptide that is obtained through cleavage ofthe BPLP protein precursor by natural maturases or prohormone convertingenzymes, or related mono or paired basic amino acid-cleaving enzymessuch as furin, PC convertases or PACE 4 (Seidah, 1995), for example.

The peptides of the invention include “peptide derivatives”.

The “peptides derivatives” are peptides having amino acid substitutionsfrom a parent peptide, preferably from one to two amino acidsubstitutions from a parent peptide particularly when said parentpeptide comprises less than 15 amino acids and preferably less than 10amino acids, but retaining the binding specificity and/or physiologicalactivity of the parent peptide. As used herein, “retaining the bindingspecificity of the parent peptide” means being able to bind to amonoclonal or polyclonal antibody that binds to one of the BPLPmaturation products or to the BPLP maturation product receptor with anaffinity that is at least one-tenth, more preferably at least one-half,and most preferably at least as great as that of one of the peptidesthat are maturation products of BPLP. Determination of such affinity ispreferably conducted under standard competitive binding immunoassayconditions. “Retaining the physiological activity of the parent peptide”means retaining the ability of any one of the BPLP maturation peptidesto bind and to modulate the activity of a metallo-ectopeptidase,especially NEP and/or APN, and more particularly NEP, and so to optimizethe local and systemic nociceptive, inflammatory, anti-depressant,and/or ion homeostatic responses to stress. Determining whether suchactivity is modulated is further described later in this specification.

The peptides of the invention include peptides or peptide derivativeswhich comprise, consist essentially of or consist of sequenceX1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6), wherein X1 represents H, Tyr, orCys, X2 represents Gln or Glp when X1 is H, or X2 represents Gln when X1is Tyr or Cys. When the peptide of the invention comprises or consistsessentially of sequence X1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6), saidsequence is the C-terminal part of the peptide of the invention.

Preferred peptides according to the invention comprise, consistessentially of, or consist of sequence QRFSR (SEQ ID NO: 3).

More particularly a peptide of the invention is the peptide thatconsists of sequence QRFSR (SEQ ID No. 3).

Another peptide of the invention is the peptide that consists ofsequence YQRFSR (SEQ ID No. 4).

Still another peptide of the invention is the peptide that consists ofsequence CQRFSR (SEQ ID NO: 5).

Yet another peptide of the invention is the peptide that consists ofsequence GlpRFSR (SEQ ID NO: 7).

Throughout the text,

Glp is pyroglutamate,

Tyr or Y is Tyrosine,

Gln or Q is glutamine,

Arg or R is Arginine,

Phe or F is Phenylalanine,

Ser or S is Serine,

Cys or C for Cystine.

The peptides according to the present invention may be prepared in aconventional manner by peptide synthesis in liquid or solid phase bysuccessive couplings of the different amino acid residues to beincorporated (from the N-terminal end to the C-terminal end in liquidphase, or from the C-terminal end to the N-terminal end in solid phase)wherein the N-terminal ends and the reactive side chains are previouslyblocked by conventional groups.

For solid phase synthesis the technique described by Merrifield may beused in particular. Alternatively, the technique described by Houbenweylin 1974 may also be used.

For more details, reference may be made to WO 98/37100.

The peptides according to the present invention may also be obtainedusing genetic engineering methods.

Preferred mimetics, including peptidomimetics, retain the bindingspecificity and/or physiological activity of the parent peptideincluding peptide derivative, as described above. As used herein, a“mimetic” is a molecule that mimics some properties of the naturalpeptides, preferably their binding specificity and physiologicalactivity. Preferred mimetics are obtained by structural modification ofpeptides according to the invention, preferably using unnatural aminoacids, D aminoacid instead of L aminoacid, conformational restraints,isosteric replacement, cyclization, or other modifications. Otherpreferred modifications include without limitation, those in which oneor more amide bond is replaced by a non-amide bond, and/or one or moreamino acid side chain is replaced by a different chemical moiety, or oneor more of the N-terminus, the C-terminus or one or more side chain isprotected by a protecting group, and/or double bonds and/or cyclizationand/or stereospecificity is introduced into the amino acid chain toincrease rigidity and/or binding affinity.

Based on the crystal structure of the binding domain of themetallo-ectopeptidase targeted by the peptide of the invention, mimeticscan also be obtained by means of computer-assisted drug designdevelopment (Oefner et al. (2000); Gomeni et al. (2001); Jones et al.(2002); Kan (2002)).

Still other preferred modifications include those intented to enhanceresistance to enzymatic degradation, improvement in the bioavailabilityin particular by nervous and gonad tissues and more generally in thepharmacokinetic properties and especially comprise:

-   -   protecting the NH₂ and COOH hydrophilic groups by esterification        (COOH) with lipophilic alcohols or by amidation (COOH) and/or by        acetylation (NH₂) or added carboxyalkyl or aromatic hydrophobic        chain at the NH₂ terminus;    -   retroinversion isomers of the CO—NH amide bonds or methylation        (or ketomethylene, methyleneoxy, hydroxyethylene) of the amide        functions;    -   substitution of L aminoacids for D aminoacids.

All of these variations are well known in the art. Thus, given thepeptide sequences disclosed herein, those skilled in the art are enabledto design and produce mimetics having binding characteristics and/orphysiological activities similar to or superior to such peptides (seee.g., Horwell et al., (1996); Liskamp et al., (1994); Gante et al.,(1994); Seebach et al., (1996)).

As used herein, the term “BPLP-peptide” refers to BPLP protein, peptidesderived from BPLP, BPLP maturation peptides, and peptides derivativesand mimetics, including peptidomimetics, of the invention.

The invention also relates to a molecular complex comprising:

-   -   a metallo-ectopeptidase receptor, especially a NEP receptor or        an APN receptor, especially a NEP receptor, binding site of the        BPLP-protein or maturation products thereof, e.g. QRFSR (SEQ ID        NO: 3);    -   the BPLP-protein or maturation products thereof, e.g. QRFSR (SEQ        ID NO: 3).

Nucleic Acids, Methods of Expression and Methods of Detection

The nucleic acids, also named polynucleotides, such as DNA or RNAmolecules, that encode the peptides, including peptides derivatives,defined above are also part of the invention, while taking into accountthe degeneration of the genetic code.

Accordingly, the present invention provides nucleic acids coding forpeptides derived from human BPLP protein, and peptides derivativesthereof.

Particularly, the present invention provides nucleic acids coding forpeptides which comprise, consist essentially of, or consist of sequenceX1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6) as above defined. When the peptideof the invention comprises or consists essentially of sequenceX1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6), said sequence is the C-terminalpart of the peptide of the invention. In preferred embodiments, thepresent invention provides acid nucleic coding for peptides whichcomprise, consist essentially of, or consist of sequence QRFSR (SEQ IDNO: 3). In a most preferred embodiment, the present invention provides anucleic acid coding for QRFSR (SEQ ID NO: 3) or a nucleic acid codingfor YQRFSR (SEQ ID NO: 4).

The nucleic acids of the invention include sequences that arehybridizable to any of the above sequences or their complementarysequences under standard hybridization conditions, preferably conditionsof high stringency.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, when a single stranded form of the nucleic acid molecule cananneal to the other nucleic acid molecule under the appropriateconditions of temperature and solution ionic strength (see Sambrook etal., 1989). The conditions of temperature and ionic strength determinethe “stringency” of the hybridization. For preliminary screening forhomologous nucleic acids, low stringency hybridization conditions,corresponding to a Tm (melting temperature) of 55° C., can be used,e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide,5×SSC, 0.5% SDS). Moderate stringency hybridization conditionscorrespond to a higher Tm, e.g., 40% formamide, with 5× or 6×SCC. Highstringency hybridization conditions correspond to the highest Tm, e.g.,50% formamide, 5× or 6×SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate.Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of Tm for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherTm) of nucleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating Tm have been derived (see Sambrooket al., supra, 9.50-9.51). For hybridization with shorter nucleic acids,i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.7-11.8). A minimum lengthfor a hybridizable nucleic acid is at least about 10 nucleotides;preferably at least about 15 nucleotides.

In a specific embodiment, the term “standard hybridization conditions”refers to a Tm of 55° C., and utilizes conditions as set forth above. Ina preferred embodiment, the Tm is 60° C. In a more preferred embodiment,the Tm is 65° C. In a specific embodiment, “high stringency” refers tohybridization and/or washing conditions at 68° C. in 0.2×SSC, at 42° C.in 50% formamide, 4×SSC, or under conditions that afford levels ofhybridization equivalent to those observed under either of these twoconditions.

The present invention further relates to vectors for cloning and/orexpression comprising a nucleic acid sequence of the invention and tohost cell comprising the nucleic acid of the invention or said vector,i.e. a host cell wherein at least one of these vectors was transferred.The expression vector according to the invention comprises a nucleicacid sequence encoding a peptide, including a peptide derivative, orprotein of the invention, said nucleic acid sequence being operablylinked to elements allowing its expression. Said vector advantageouslycontains a promoter sequence, signals for initiation and termination oftranslation, as well as appropriate regions for regulation oftranslation. Its insertion into the host cell may be transient orstable. Said vector may also contain specific signals for secretion ofthe translated protein.

These various control signals are selected according to the host celland may be inserted into vectors which self-replicate in the selectedhost cell, or into vectors which integrate the genome of said host.

Host cells may be prokaryotic or eukaryotic, including but not limitedto bacteria, yeasts, plant cells, insect cells, mammalian cells,including cell lines which are commercially available. Preferredexamples for host cells are COS-1, HEK cells, 293 cells, or CHO cells.

A subject of the present invention is also a method for producing arecombinant BPLP-peptide, wherein said host cell is transfected withsaid expression vector and is cultured under conditions allowing theexpression of a BPLP-peptide. The transfection of the host cell may beperformed using any standard technique, such as electroporation orphosphate calcium precipitation or Lipofectine®.

The protein or peptide can then be collected and purified, by means ofwell-known procedures for purification: the recombinant peptide orprotein may be purified from lysates or cell extracts, from thesupernatant of the culture medium, by methods such as HPLCchromatography, immunoaffinity techniques with specific antibodies, andthe like.

The present invention further relates to methods of in vitro prognosisand/or diagnosis wherein the nucleic acid sequences of the invention orprobes or primers derived thereof are used to detect aberrant synthesis,including abnormal high or low synthesis, or genetic abnormalities atthe BPLP gene level.

The invention thus provides an in vitro method for prognosis and/ordiagnosis of a condition involving an altered production of BPLP or ofany of its maturation products, which method comprises detecting in abiological sample of a test subject, an abnormality in terms of qualityand/or quantity in the BPLP gene or in its transcript.

The term “prognosis” refers to the determination or confirmation of alikelihood of a disease or condition to arise.

The present invention is more particularly directed to a method fordetecting an abnormality in the BPLP gene comprising the steps of:

-   -   contacting a biological sample containing DNA with specific        oligonucleotides permitting the amplification of all or part of        the BPLP gene, the DNA contained in the sample having being        rendered accessible, where appropriate, to hybridization, and        under conditions permitting a hybridization of the primers with        the DNA contained in the biological sample;    -   amplifying said DNA;    -   detecting the amplification products;    -   comparing the amplified products as obtained to the amplified        products obtained with a normal control biological sample, and        thereby detecting a possible abnormality in the BPLP gene.

The method of the invention can also be applied to the detection of anabnormality in the transcript of the BPLP gene, by amplifying the mRNAscontained in a biological sample, for example by RT-PCR.

Thus another subject of the present invention is a method for detectingan abnormality in the BPLP transcript, as previously defined comprisingthe steps of:

-   -   producing cDNA from mRNA contained in a biological sample;        -   contacting said cDNA with specific oligonucleotides            permitting the amplification of all or part of the            transcript of the BPLP gene, under conditions permitting a            hybridization of the primers with said cDNA;    -   amplifying said cDNA;    -   detecting the amplification products;    -   comparing the amplified products as obtained to the amplified        products obtained with a normal control biological sample, and        thereby detecting a possible abnormality in the transcript of        the BPLP gene.

This comparison of the amplified products obtained from the biologicalsample with the amplified products obtained with a normal biologicalsample is a quantitative comparison and/or a qualitative comparison. Inthis latter case, comparison can be carried out for example by specificprobe hybridization, by sequencing or by restriction site analysis.

One skilled in the art very well knows the standard methods foranalysing the DNA contained in a biological sample and for diagnosing agenetic disorder. Many strategies for genotypic analysis are available.

Preferably, one can use the DGGE method (Denaturing Gradient GelElectrophoresis), or the SSCP method (Single Strand ConformationPolymorphism) for detecting an abnormality in the BPLP gene. Suchmethods are preferably followed by direct sequencing. The RT-PCR methodmay be advantageously used for detecting abnormalities in the BPLPtranscript, as it allows to visualize the consequences of a splicingmutation such as exon skipping or aberrant splicing due to theactivation of a cryptic site. This method is preferably followed bydirect sequencing as well. The more recently developed technique usingDNA chip can also be advantageously implemented for detecting anabnormality in the BPLP gene.

These methods for detecting an abnormality in the BPLP gene, or in itstranscript, are particularly useful to identify mutations that result innonfunctional BPLP protein or maturation products, and are advantageousfor in vitro prognosis and/or diagnosis of diseases, wherein the BPLPgene is involved.

Examples of such diseases are diseases cited in the “therapeuticapplication” section.

Antibodies and Methods of Detection

The present invention further provides antibodies, specifically directedagainst (i.e. that specifically recognizes) the BPLP protein. Thepresent invention further provides antibodies, specifically directedagainst (i.e. that specifically recognizes) the peptides as abovedefined, including peptides derivatives.

Accordingly, the present invention provides antibodies directed againstpeptides derived from human BPLP protein, and peptide derivativesthereof.

More particularly, the present invention provides antibodies directedagainst peptides which comprise, consist essentially of, or consist ofsequence X1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6) as above defined. When thepeptide of the invention comprises or consists essentially of sequenceX1-X2-Arg-Phe-Ser-Arg (SEQ ID NO: 6), said sequence is the C-terminalpart of the peptide of the invention. In preferred embodiments, thepresent invention provides antibodies directed against (i.e. thatspecifically recognize) peptides which comprise, consist essentially of,or consist of sequence QRFSR (SEQ ID NO: 3). In a most preferredembodiment, the present invention provides antibodies directed against(i.e. that specifically recognize) QRFSR (SEQ ID NO: 3) or antibodiesdirected against (i.e. that specifically recognize) YQRFSR (SEQ ID NO:4) or antibodies directed against (i.e. that specifically recognize)CQRFSR (SEQ ID NO: 5).

The term “antibody” in its various grammatical forms is used herein torefer to immunoglobulin molecules and immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope. Exemplary antibody molecules are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand portions of an immunoglobulin molecule, including those portionsknown in the art as Fab, Fab′, F(ab′)2 and F(v).

Antibodies that inhibit the interaction of a BPLP maturation product ora peptide derivative thereof with its receptor are more particularlyuseful.

Whereas polyclonal antibodies may be used, monoclonal antibodies arepreferred for they are more reproducible in the long run.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpeptide, including conjugate peptide, of the present inventionsubcutaneously to New Zealand white rabbits which have first been bledto obtain pre-immune serum. The antigens can be injected at a totalvolume of 50 μl per site at ten different sites or at least fivedifferent sites. The rabbits are then bled five weeks after the firstinjection and periodically boosted with the same antigen administeredsubcutaneously at five fold lower concentration than the primaryinjection at maximum depending on quality of the immune response threetimes every six weeks. A sample of serum is then collected every 10 daysafter each boost. Polyclonal antibodies are then recovered from theserum by affinity chromatography using the corresponding antigen tocapture the antibody. This and other procedures for raising polyclonalantibodies are disclosed in E. Harlow, et. al., editors, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory, New York (1988).

A “monoclonal antibody” in its various grammatical forms refers to apopulation of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g. a bispecific monoclonal antibody.

Laboratory methods for preparing monoclonal antibodies are well known inthe art (see, for example, Harlow et al., supra). Monoclonal antibodies(Mabs) may be prepared by immunizing a mammal, e.g. a mouse, rat,rabbit, goat, human and the like, against the purified BPLP protein,BPLP maturation products or peptide derivatives thereof, includingconjugated BPLP-peptides. The antibody-producing cells in the immunizedmammal are isolated and fused with myeloma or heteromyeloma cells toproduce hybrid cells (hybridoma). The hybridoma cells producing themonoclonal antibodies are used as a source of the desired monoclonalantibody.

While Mabs can be produced by hybridoma culture, the invention is not tobe so limited. Also contemplated is the use of Mabs produced by anexpressing nucleic acid cloned from a hybridoma. That is, the nucleicacid expressing the molecules secreted by a hybridoma can be transferredinto another cell line to produce a transformant. The transformant isgenotypically distinct from the original hybridoma but is also capableof producing antibody molecules of this invention, includingimmunologically active fragments of whole antibody molecules,corresponding to those secreted by the hybridoma. In addition, theliterature provides methods for forming chimeric antibodies, humanizedantibodies, single chain antibodies and the like variations on a basicimmunoreactive antibody fragment. All of these are considered within thescope of the invention insofar as a class and specificity of antibody isdisclosed and claimed, regardless of the precise variant structure thatone skilled in the art may construct.

The present invention further relates to an in vitro method fordiagnosis, prognosis or determination of the evolution of a conditioninvolving an altered production (i.e. a decrease or an increase ofproduction in comparison to a control subject) of BPLP or of any of itsmaturation products. The method comprises detecting, or quantifying in abiological sample of a test subject, a BPLP protein or maturationproducts thereof, especially QRFSR (SEQ ID NO: 3), compared with thesame in a biological sample of a control subject.

Examples of such conditions are diseases cited in the “therapeuticapplications” section.

A “biological sample” is a fluid from a subject, including serum, blood,spinal fluid, cerebrospinal fluid, urine, milk, saliva or a tissueextract or a tissue or organ biopsy such as brain, spinal cord, bonetissue, kidney, prostate, placenta, dental tissue, glandular mucosa ofstomach, intestine, salivary gland tissue, mammary glands, for example.

“A subject” or “a patient” is a vertebrate, e.g. a mammal, preferably ahuman being, regardless of his/her age, sex and general condition.Children and infants are also encompassed. The test subject may beasymptomatic, may be considered likely to develop the disease orcondition. Subjects with a suspicion of a target disorder or subjectswho have already shown symptoms of the disease or condition can also betested.

The “control subject” may be a healthy subject or a subject without anyapparent disorder that can involve the BPLP protein or one of itsmaturation products. In order to determine the evolution of a conditioninvolving the BPLP protein or one of its maturation products, it may bevery useful to test a subject for the expression of BPLP protein or amaturation products thereof, and to monitor the effect of a drug or thespreading of the condition, by testing him/her a second time, e.g. a fewweeks later. In that case the results of the second test are comparedwith the results of the first test, and in general also with the resultsobtained with a “healthy” subject. The “control subject” then referseither to the same test subject or to a “healthy subject”.

The term “diagnosis” refers to the determination or the confirmation ofa disease or condition in a subject.

The term “prognosis” refers to the determination or confirmation of alikelihood of a disease or condition to arise.

The “expression or production of a BPLP protein or a maturation productthereof” may be determined by assaying the BPLP protein or a maturationproducts thereof.

Such assay methods comprise contacting a biological sample with abinding partner capable of selectively interacting with a BPLP proteinor maturation products thereof, especially QRFSR (SEQ ID NO: 3), presentin the sample. The binding partner is generally an antibody, that may bepolyclonal or monoclonal, preferably monoclonal.

Methods for producing antibodies as described above in accordance withtherapy can also be easily adapted to produce antibodies useful for thediagnostic or prognostic methods according to the invention.

For example, the presence or production of BPLP protein or of any of itsmaturation products, or a mutated form of the protein or of thematuration product, can be detected by incubating a biological samplewith an antibody that specifically recognizes the BPLP protein or anantibody that specifically recognizes a maturation product thereof,especially QRFSR (SEQ ID NO: 3), e.g. using standard electrophoretic andliquid or solid immunodiagnostic techniques, including immunoassays suchas competition, direct reaction, or sandwich type assays. Such assaysinclude, but are not limited to, Western blots; agglutination tests;enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidintype assays; radioimmunoassay such as those using radioiodinated ortritiated BPLP protein or any of its maturation products, especiallyQRFSR (SEQ ID NO: 3); immunoelectrophoresis; immunoprecipitation, etc.The reactions generally include revealing labels such as fluorescent,chemiluminescent, radioactive, enzymatic labels or dye molecules, orother methods for detecting the formation of a complex between theantigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound BPLPprotein or unbound maturation products thereof, especially unbound QRFSR(SEQ ID NO: 3), from the bound BPLP protein or maturation products,especially QHNPR (SEQ ID NO: 8), to the specific antibody which isimmobilized on a solid phase. Solid supports which can be used in thepractice of the invention include supports such as nitrocellulose (e.g.,in membrane or microtiter well form); polyvinylchloride (e.g., sheets ormicrotiter wells); polystyrene latex (e.g., beads or microtiter plates);polyvinylidine fluoride; diazotized paper; nylon membranes; activatedbeads, magnetically responsive beads, and the like.

Thus, in one particular embodiment, the presence of bound BPLP proteinor maturation products thereof, especially QRFSR (SEQ ID NO: 3), from abiological sample can be readily detected using a secondary bindercomprising another antibody, that can be readily conjugated to adetectable enzyme label, such as horseradish peroxidase, alkalinephosphatase or urease, using methods known to those of skill in the art.An appropriate enzyme substrate is then used to generate a detectablesignal, such as a chromogenic or fluorogenic signal for example. Inother related embodiments, competitive-type ELISA techniques can bepracticed using methods known to those skilled in the art.

The above-described assay reagents, including the antibodies, can beprovided in kits, with suitable instructions and other necessaryreagents, in order to conduct immunoassays as described above. The kitcan also contain, depending on the particular immunoassay used, suitablelabels and other packaged reagents and materials (i.e. wash buffers andthe like). Standard immunoassays, such as those described above, can beconducted using these kits.

Gene Therapy

In accordance with the present invention, the modulation of the membranemetallopeptidase activity may be achieved by modifying (i.e. increasingor decreasing) the amount of BPLP protein, maturation products thereofin the cells of a patient and release therefrom, or expressing andpossibly releasing a peptide, including peptide derivative, as definedabove. Increasing of the amount of BPLP protein or maturation productsthereof in the cells of a patient and possibly release therefrom,expressing and possibly releasing a peptide as defined above including apeptide derivative, may be performed by transfecting the cells with BPLPexpressing vector or a vector that expresses a BPLP protein, a BPLPmaturation product or a peptide as defined above, including a peptidederivative, e.g. in the form of a naked DNA or as a viral vector.

Preferably, the nucleic acid of this invention forms part of a vector.Such vector is a nucleic acid comprising a coding sequence operativelyassociated with sequences that control expression of the protein orpeptide in a cell transfected with the vector.

The use of such a vector indeed makes it possible to improve theadministration of the nucleic acid into the cells of the subject andespecially to the cells to be treated, and also to increase itsstability in the said cells, which makes it possible to obtain a durabletherapeutic effect. Furthermore, it is possible to introduce severalnucleic acid sequences into the same vector, which also increases theefficacy of the treatment.

The vector used may be of diverse origin, as long as it is capable oftransforming animal cells, preferably human cells. In a preferredembodiment of the invention, a viral vector is used which can be chosenfrom adenoviruses, retroviruses, adeno-associated viruses (AAV),lentivirus, herpes virus, cytomegalovirus (CMV), vaccinia virus and thelike. Vectors derived from adenoviruses, retroviruses or AAVs,HIV-derived retroviral vectors, incorporating heterologous nucleic acidsequences have been described in the literature.

The present invention therefore also relates to any recombinant viruscomprising, inserted into its genome, nucleic acid sequence that encodesthe BPLP protein, a BPLP maturation product or a peptide as definedabove, including a peptide derivative.

Advantageously, the recombinant virus according to the invention is adefective virus, devoid of at least the sequences necessary for thereplication of the said virus in the infected cell.

It is particularly advantageous to use the nucleic acid sequences of theinvention in a form incorporated in an adenovirus, an AAV or a defectiverecombinant retrovirus.

Targeted gene delivery is described in International Pat. Publication WO95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. Forthe past decade, there has been increasing use of liposomes forencapsulation and transfection of nucleic acids in vitro. Informationregarding liposome is provided in the “pharmaceutical composition”section of the present application as well.

It is also possible to introduce the vector in vivo as a naked DNAplasmid. Naked DNA vectors for gene therapy can be introduced into thedesired host cells by methods known in the art, e.g., transfection,electroporation, microinjection, transduction, cell fusion, DEAEdextran, calcium phosphate precipitation, Lipofectamine®, use of a genegun, or use of a DNA vector transporter.

Pharmaceutical Compositions

The BPLP-peptides (that is to say the BPLP protein, peptides derivedfrom BPLP, maturation products, peptides defined above, includingpeptide derivatives and mimetics), or the nucleic acids that encode suchBPLP-peptides and antibodies against said BPLP-peptides can beformulated in pharmaceutical compositions in association with apharmaceutically acceptable carrier. For instance the pharmaceuticalcompositions are suitable for a topical, oral, sublingual, parenteral,intranasal, intravenous, intramuscular, subcutaneous, transcutaneous orintraocular administration and the like.

A subject matter of the invention is also a pharmaceutical compositioncomprising a polymer of said BPLP-peptide or mimetic thereof.

Preferably, the nucleic acid forms part of a vector expressing saidnucleic acid.

Preferably, the pharmaceutical compositions contain vehicles which arepharmaceutically acceptable for a formulation capable of being injected.

The suitable pharmaceutical compositions may be in particular isotonic,sterile, saline solutions (monosodium or disodium phosphate, sodium,potassium, calcium or magnesium chloride and the like or mixtures ofsuch salts), or dry, especially freeze-dried compositions which uponaddition, depending on the case, of sterilized water or physiologicalsaline, permit the constitution of injectable solutions.

The doses of BPLP-peptide, antibodies or nucleic acid used for theadministration can be adapted as a function of various parameters, andin particular as a function of the mode of administration used, of therelevant pathology, or alternatively of the desired duration oftreatment.

To prepare pharmaceutical compositions for peptide therapy, an effectiveamount of the BPLP-peptide may be dissolved or dispersed in apharmaceutically acceptable carrier or aqueous medium.

Examples of pharmaceutical formulations are provided hereafter.

Pharmaceutical compositions comprise an effective amount of theBPLP-peptide, nucleic acid or antibodies, in a pharmaceuticallyacceptable carrier or aqueous medium.

“Pharmaceutically” or “pharmaceutically acceptable” refer to molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, including ahuman, as appropriate.

As used herein, a “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetables oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminiummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with avariety of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms, such as the type of injectable solutions described above,but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, sterile aqueous media which can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage could be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion, (see for example, “Remington's PharmaceuticalSciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variationin dosage will necessarily occur depending on the condition of thesubject being treated. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The BPLP-peptide of interest may be formulated within a therapeuticmixture to comprise about 0.0001 to 100 milligrams, or about 0.001 to0.1 milligrams, or about 0.1 to 1.0 or even about 1 milligram to 10milligrams or even about 10 to 100 milligrams per dose or so. Multipledoses can also be administered. Preferred dosages are from about 0.1μg/kg to about 1 mg/kg, more preferably from about 1 μg/kg to about 100μg/kg, and most preferably from about 10 μg/kg to about 100 μg/kg.

In addition to the formulations for parenteral administration, such asintravenous or intramuscular injection, other pharmaceuticallyacceptable forms include, e.g. tablets or other solids for oraladministration; liposomal formulations; time release capsules; and anyother form currently used, including creams.

Other routes of administration are contemplated, including nasalsolutions or sprays, aerosols or inhalants, or vaginal or rectalsuppositories and pessaries or cream, and long-acting delivery polymers.

In certain embodiments, the use of liposomes and/or nanoparticles iscontemplated for the introduction of BPLP-peptide agents, as well asnucleic acid vectors or antibodies into host cells.

The invention also relates to pharmaceutical compositions above definedfurther comprising a second pharmaceutical agent that actssynergistically with BPLP-peptide.

Therapeutic Applications

The BPLP-peptides described above, antibodies against said BPLP-peptidesor nucleic acids coding for said BPLP-peptides are useful in theprevention or treatment of diseases or disorders, wherein a modulationof the activity of a membrane metallo-ectopeptidase is sought, moreparticularly a membrane-zinc metallopeptidase, such as NEP and APN.

Natural NEP substrates are mainly the peptide hormones: Enkephalins,Substance P, Bradykinin, Angiotensin II and Atrial Natriuretic Peptidewhich play key role in the control of central and peripheral painperception, inflammatory phenomena, mineral exchange and/or arterialtone (Rogues et al., 1993).

More particularly, neutral endopeptidase, NEP 24-11, is distributed bothin nervous and peripheral tissues of mammals, and in the periphery it isparticularly abundant in the kidney and placenta. In these tissues thecell-surface metallopeptidase NEP participates in the postsecretoryprocessing and metabolism of neuropeptides, systemic immunoregulatorypeptides and peptide-hormones. By controlling the active levels ofcirculating or secreted regulatory peptides, NEP modulates theirphysiological receptor-mediated action. Hence, the membrane-anchored NEPis involved in regulating the activity of: potent vasoactive peptidessuch as Substance P, Bradykinin (BK), Atrial Natriuretic peptide (ANP),and Angiotensin II (AII); potent inflammatory/immunoregulatory peptidessuch as Substance P and BK and fMet-Leu-Phe (fMLP); potent opioidneuropeptides such as Met and Leu-Enkephalins (Enk) and potent mineralexchange and fluid homeostasis regulatory peptides such as ANP, C-typeNatriuretic Peptide (CNP) and B-type Natriuretic Peptide (BNP). Howeverthe levels of these peptides are changed through the NEP-inducedformation/degradation only in regions where they are tonically releasedor where their release is triggered by a stimulus.

From an integrative point of view, the NEP biological activity is tocontrol the active levels of peptidergic signals involved in arterialtension regulation, in inflammatory phenomena and in water-mineralhomeostasis, as well as, in the control of pain processing. From aclinical point of view, this substantiates the fact that NEP is animportant drug target in various disease states. For example, byinhibiting NEP, thereby increasing the levels and duration of action ofcentral or peripheral endogenous opioids, an analgesic effect or ananti-depressant effect could be obtained, or by inhibiting endogenousAII formation and substance P, BK and ANP inactivation,antihypertensive, natriuretic and diuretic agents could be obtained. Themain advantage of modifying the concentrations of endogenous peptides byuse of NEP inhibitors is that the pharmacological effects are inducedonly at receptor stimulated by the natural effectors, and are criticallydependent on the tonic or stimulus-evoked release of the naturaleffectors happening upon environmental, behavioral andphysiopathological stressful situations (Rogues et al, 1993).

Examples of mammalian membrane metallopeptidases besides NEP are ECE(Endothelin-Converting Enzymes), in particular ECE1 and ECE2, theerythrocyte cell-surface antigen KELL and the product of PEX geneassociated with X-linked hypophosphatemic rickets, as well as ACE(Angiotensin Converting Enzyme) and APN (Aminopeptidase N).

Inhibition of ACE and/or ECE has a significant application in thetreatment of hypertension and the prevention and treatment ofatherosclerosis.

Inhibition of APN in conjunction with NEP has significant application inthe treatment of pain and depression.

Inhibition of related membrane metallopeptidases has therapeutic effectsin the treatment of tumors, namely ovarian, colorectal, brain, lung,pancreas, gastric and melanoma cancers, and reducing the incidence ofmetastasis, atherosclerosis and/or hypertension. Inhibitions of relatedmembrane metallopeptidases has also therapeutic effects in paincontrolling. Such antinociceptive effects on acute pain are analgesiceffects but also effects on chronic inflammatory pain such as arthritisor inflammatory bowel disease.

Furthermore, inhibition of bacterial or viral metallopeptidase isexpected to have anti-infection effects.

Metallopeptidases playing an important role in pathogen host tissueinvasion and immunological and inflammatory processes, for example thoseof Streptococcus pyogenes, Pseudomonas aeruginosa, Porphyromonasgingivalis and Legionella pneumophila.

Furthermore, bacterial metallopeptidases, especiallyzinc-metallopeptidases play an important role in the diseases caused byproteolytic toxins, such as the toxins of B. anthracis (Anthrax Lethalfactor) and the neurotoxins of C. tetanum and botulinum.

Other metallopeptidases play an important role in various infectionssuch as infections caused by HIV (FR 2 707 169).

The importance of proteinase inhibitors for the treatment of bacterialor viral diseases may be found in J. Potempa and J. Travis.

The different roles of metallopeptidases are disclosed in Turner et al,2001; Kenny et al, 1977; Kenny et al, 1987; Beaumont et al, 1996.

One object of the present invention is the use of the above describedtherapeutic peptides or nucleic acids as analgesic agents oranti-depressant agents by inhibiting NEP and APN at peripheral, spinaland/or supraspinal levels and thereby increasing the levels and durationof action of central or peripheral endogenous opioids, includingenkephalins.

The prevention or treatment of pain, especially acute and chronic pain,visceral inflammatory and neuropathic pain, is contemplated.

The prevention or treatment of any hydro-mineral imbalance is also anaim of the invention. Among target disorders one may cite bone, teeth,kidney, parathyroid, pancreas, intestine, stomach mucosa, prostate, andsalivary gland disorders that are caused by hydro-mineral imbalance.

In particular, the disorder may be selected from the group consisting ofhyper or hypo-parathyroidism, osteoporosis, pancreatitis, submandibulargland lithiasis, nephrolithiasis and osteodystrophy.

The prevention or treatment of impaired interpersonal and behaviouraldisorders is of further interest. Various mental disorders are describedin WO 02/051434.

In particular the invention is drawn at any disorder selected from thegroup consisting of avoidance disorder, decreased awareness disorder,autistic disorder, attention deficit hyperactivity disorder, arousaldisorder, hospitalism, impaired interpersonal functioning andrelationship to the external world, schizoid personality disorder,schizophrenia, depressive disorder, decreased interest in environment,impaired social activity linked to sexuality, and impaired sexualbehaviour, including untimely ejaculation and hyperactive sexual.

Diseases wherein a modulation of a membrane metallopeptidase is soughtalso include hypertension, aterosclerosis, tumor, inflammatory arthritisand bowel disease.

Treatment of infections is also encompassed. Especially, the importanceof proteinase inhibitors for the treatment of bacterial or viraldiseases may be found in J. Potempa and Travis.

The BPLP-peptides, antibodies or nucleic acids described above are alsouseful for controlling immuno-inflammatory responses.

The BPLP-peptides, antibodies or nucleic acids as defined above are alsouseful as a natriuretic agent or a diuretic agent.

Another object of the present invention is the use of the abovedescribed peptides or nucleic acids as a substitute in the treatment ofdrug abuse, notably morphine drug abuse.

Indeed, studies have suggested that the vulnerability to drug abuse andthe development of reward and drug dependence is at least in part, aresult of pre-existent or induced modifications and/or defect of theendogenous opioid system. In this regard, using BPLP-peptide or nucleicacid to potentiate the effects of endogenous enkephalins will reduce thevarious side-effects (somatic signs of withdrawal) produced byinterruption of chronic morphine or heroin administration.

According to the invention, reducing the inhibitory effect of theBPLP-peptides on NEP may be desired, e.g. by using an antibody againstthe BPLP protein or peptides. This enhancement of NEP activity isparticularly advantageous in the treatment of neurodegenerative diseasessuch as a disease or disorder associated with amyloidosis. Indeed it hasbeen shown that inhibitors of neprilysin (a neutral endopeptidase, NEPor enkephalinase) by synthetic inhibitor, raises amyloid p levels(Newell et al, 2003). Leissring et al, 2003 further reported thattransgenic overexpression of neprilysin in neurons significantly reducesbrain Aβ levels, retards or completely prevents amyloid plaque formationand its associated cytopathology, and rescues the premature lethalitypresent in amyloid precursor protein transgenic mice.

A disease or disorder is associated with amyloidosis when amyloiddeposits or amyloid plaques are found in or in proximity to tissuesaffected by the disease, or when the disease is characterized byoverproduction of a protein that is or can become insoluble. The amyloidplaques may provoke pathological effects directly or indirectly by knownor unknown mechanisms. Examples of amyloid diseases include, but are notlimited to, systemic diseases, such as chronic inflammatory illnesses,multiple myeloma, macroglobulinemia, familial amyloid polyneuropathy(Portuguese) and cardiomyopathy (Danish), systemic senile amyloidosis,familial amyloid polynephropathy (Iowa), familial amyloidosis (Finnish),Gerstmann-Straussler-Scheinker syndrome, familial amyloid nephropathywith urticaria and deafness (Muckle-Wells syndrome), medullary carcinomaof thyroid, isolated atrial amyloid, and hemodialysis-associatedamyloidosis (HAA); and neurodegenerative diseases.

The term “neurodegenerative disease” refers to a disease or disorder ofthe nervous system, particularly involving the brain, that manifestswith symptoms characteristic of brain or nerve dysfunction, e.g.,short-term or long-term memory lapse or defects, dementia, cognitiondefects, balance and coordination problems, and emotional and behavioraldeficiencies. The present invention is more particularly concerned withneurodegenerative diseases that are associated with amyloidosis. Suchdiseases are “associated with amyloidosis” when histopathological(biopsy) samples of brain tissue from subjects who demonstrate suchsymptoms would reveal amyloid plaque formation. As biopsy samples frombrain, especially human brain, are obtained with great difficulty fromliving subjects or might not be available at all, often the associationof a symptom or symptoms of neurodegenerative disease with amyloidosisis based on criteria other than the presence of amyloid deposits, suchas plaques or fibrils, in a biopsy sample.

In a specific embodiment, according to the present invention theneurodegenerative disease is Alzheimer's disease (AD). In otherembodiments, the disease may be the rare Swedish disease characterizedby a double KM to NL mutation in amyloid precursor protein (APP) nearthe amino-terminus of the βAP portion of APP. Another such disease ishereditary cerebral hemorrhage with amyloidosis (HCHA or HCHWA)-Dutchtype. Other such diseases known in the art and within the scope of thepresent invention include, but are not limited to, sporadic cerebralamyloid angiopathy, hereditary cerebral amyloid angiopathy, Down'ssyndrome, Parkinson-dementia of Guam, and age-related asymptomaticamyloid angiopathy.

In a further aspect, the neurodegenerative disease is a subacutespongiform encephalopathy, such as but not limited to, scrapie,Creutzfeldt-Jakob disease, Gerstmann-Straussler disease, kuru, chronicwasting disease of mule-deer and elk, bovine spongiform encephalopathyof cattle, and mink transmissible encephalopathy.

The invention further relates to the use of an agent that modulates theinteraction between endogenous BPLP protein or maturation product, e.g.QRFSR (SEQ ID NO: 3), and a membrane metallopeptidase for thepreparation of a therapeutic composition for preventing or treatingdiseases wherein a modulation of the activity of said membranemetallopeptidase is sought.

Screening Methods

The methods that allow a person skilled in the art to select and purifycandidate compounds that bind to the same targets and have an agonist oran antagonist biological activity of the BPLP protein or maturationproducts thereof, e.g. the QRFSR peptide (SEQ ID NO: 3), are describedhereunder.

The candidate compound may be a protein, a peptide, a hormone, anantibody or a synthetic compound which is either a peptide or a nonpeptidic molecule, such as any compound that can be synthesized by theconventional methods of organic chemistry.

The invention provides an in vitro method for screening compounds fortheir ability to bind to the NEP binding site for the BPLP protein or amaturation product thereof, e.g. the QRFSR peptide (SEQ ID NO: 3),comprising the steps of:

a) incubating a candidate compound with a NEP expressing cell, in thepresence of the BPLP protein or a maturation product thereof, e.g. theQRFSR peptide (SEQ ID NO: 3), or any peptide retaining the bindingspecificity or the physiological activity of BPLP protein or of itsmaturation products, e.g. the peptide YQRFSR (SEQ ID NO: 4);

b) determining the ability of the candidate compound to compete with theBPLP protein or a maturation product thereof, e.g. the QRFSR peptide(SEQ ID NO: 3) or with the peptide retaining the binding specificity orthe physiological activity of BPLP protein or of its maturationproducts, e.g. the peptide YQRFSR (SEQ ID NO: 4), for binding to NEP.

Binding assays of the candidate compound are generally performed at 4°C. to 25° C. or 37° C.

The NEP expressing cell may be in a cell culture, such as a confluenttarget cell culture monolayer, or a target organ specimen or a tissuesample (e.g. cryosections, slices, membrane preparations or crudehomogenates) that contains NEP binding sites for the BPLP protein or amaturation product thereof, e.g. the QRFSR peptide (SEQ ID NO: 3).

A preferred tissue sample that is used in the screening methodsaccording to the present invention is a membrane preparation or slicesof spinal cord from a mammal, a tissue known to be appropriated for NEPactivity measurement.

Other preferred tissue samples that can be used in the screening methodsaccording to the present invention are all peripheral tissuepreparations that are known to be enriched in NEP-peptidase and/or to betargets for the BPLP protein or a maturation product thereof, e.g. theQRFSR peptide (SEQ ID NO: 3). For example one may use mammal renal outermedulla, placenta, testis, prostate and bone. For example, such aprocedure can be applied to tissues and/or cells of mouse, rat or humanorigin or cell lines transfected with metallo-ectopeptidase cDNA, inparticular NEP cDNA, especially human NEP cDNA.

The BPLP-protein or maturation product thereof (or the peptide thatretains the binding specificity or the physiological activity of theBPLP protein or of its matured products) is preferably labeled, e.g. bya radioactive (³²P, ³⁵S, ³H, ¹²⁵I etc. . . . ) or non-radioactive label(digoxigenin, CyDye-europium, fluorescein etc.). It is then incubatedwith the NEP expressing cell during a time sufficient and underconditions for the specific binding to take place.

The label specifically bound to the cell may then be quantified in thepresence of various concentrations of said candidate compound, forexample from 10⁻¹⁰ to 10⁻⁵ M.

Accordingly, the present invention further provides a process forscreening a compound that specifically bind to the NEP binding sites forthe BPLP protein, or maturation product thereof comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (such as cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the BPLP protein ormaturation products thereof;

b) adding the candidate compound to be tested in competition withhalf-saturation concentration of labeled protein or maturation productthereof (or a peptide that retains the binding specificity or thephysiological activity of the BPLP protein or of its matured products);

c) incubating the cell culture, organ specimen or tissue sample of stepa) in the presence of the candidate compound during a time sufficientand under conditions for the specific binding to take place;

d) quantifying the label specifically bound to the cell culture, organspecimen or tissue sample in the presence of various concentrations ofcandidate compound (preferably 10⁻¹⁰ to 10⁻⁵ M).

In said above process, a half saturating concentration is theconcentration of the labeled BPLP protein or maturation product thereof,e.g. the QRFSR peptide (SEQ ID NO: 3) (or the peptide that retains thebinding specificity or the physiological activity of the BPLP protein orof its matured products) which binds 50% of the NEP binding sites.

This process also allows to define the relative affinity of thecandidate compound compared to the BPLP protein, or maturation products,e.g. QRFSR affinity (SEQ ID NO: 3) (or the peptide that retains thebinding specificity or the physiological activity of the BPLP protein orof its matured products).

Another object of the present invention is a process for determining therelative affinity of ligand compounds that specifically bind to the NEPbinding sites for the BPLP protein, or maturation products, (or thepeptide that retains the binding specificity or the physiologicalactivity of the BPLP protein or of its matured products), said processcomprising the steps a), b), c) and d) of the above process for eachcandidate compound and further comprising the step e) of comparing theaffinity of each candidate compound quantified in step d) to the one ofthe other candidate compounds.

Another object of the present invention is a process for determining theaffinity of a compound that specifically binds to the NEP binding sitefor the BPLP protein or maturation products thereof, comprising thesteps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (such as cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the BPLP protein ormaturation products thereof;

b) adding the candidate compound which has previously been labeled witha radioactive or a nonradioactive label;

c) incubating the cell culture, organ specimen or tissue sample of stepa) in the presence of the labeled candidate compound during a timesufficient and under conditions for the specific binding to take place;and

d) quantifying the label specifically bound to the cell culture, organspecimen or tissue sample in the presence of various concentrations ofthe labeled candidate compound (preferably 10⁻¹⁰ to 10⁻⁵ M).

The candidate compound is preferably labeled, e.g. by a radioactive(³²P, ³⁵S, ³H, ¹²⁵I etc. . . . ) or non-radioactive label (digoxigenin,CyDye-europium, fluorescein etc.). It is then incubated with the NEPexpressing cell during a time sufficient and under conditions for thespecific binding to take place.

One may further compare the affinity of each candidate compoundquantified to the one of the other candidate compounds, so that therelative affinity of candidate compound that specifically binds to theNEP binding site for the BPLP protein or a maturation product thereof,e.g. the QRFSR peptide (SEQ ID NO: 3), is determined.

The invention further provides an in vitro method for screeningcompounds for their ability to act as agonists or antagonists of theBPLP protein or maturation products thereof on NEP activity, whichmethod comprises the steps of:

a) incubating a candidate compound with a NEP expressing cell, in thepresence of (i) the BPLP protein or a maturation product thereof, e.g.the QRFSR peptide (SEQ ID NO: 3), or any peptide retaining the bindingspecificity or the physiological activity of the BPLP protein or of itsmatured products, and (ii) a NEP substrate;

b) determining the endoproteolysis of the NEP substrate by the NEP,wherein an increased endoproteolysis in the presence of the candidatecompound, in comparison with the endoproteolysis in the absence of thecandidate compound, is indicative of an antagonist activity; while adecreased endoproteolysis in the presence of the candidate compound, incomparison with the endoproteolysis in the absence of the candidatecompound, is indicative of an agonist activity.

As used herein, an agonist of a BPLP protein or maturation productthereof is a molecule which has the ability to inhibit ametallo-ectopeptidase activity, especially NEP or APN activity.

As used herein, an antagonist of a BPLP protein or maturation productthereof is a molecule which has the ability to increase ametallo-peptidase activity, especially NEP or APN activity.

Furthermore, the agonist or antagonist activity of the candidatecompound can be assessed in determining the metabolic changes induced bythis candidate compound on its target, such as the synthesis and/orrelease of the primary or secondary messenger metabolites as a result ofa transduction signal via the protein kinases or adenylate cyclase andthe activation of a protein of the G family.

In particular embodiments, the present invention also pertains to aprocess for screening a compound that is an agonist of the BPLP proteinor a maturation product thereof, comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (such as cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the BPLP protein or amaturation product thereof;

b) incubating the cell culture, organ specimen or tissue sample of stepa) at concentrations allowing measurement of NEP enzymatic activity inthe presence of the candidate compound (preferably 10⁻¹⁰ to 10⁻⁵ M), ahalf-saturating concentration of the BPLP protein or a maturationproduct thereof (or any peptide retaining the binding specificity or thephysiological activity of the BPLP protein or of its matured products)and a NEP substrate during a time sufficient for the endoproteolysis ofthe NEP substrate to take place under initial velocity conditions;

c) quantifying the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrateendoproteolysis, respectively in the presence or in the absence of thecandidate compound and in the presence or in the absence of the BPLPprotein or a maturation product thereof, or the peptide retaining thebinding specificity or the physiological activity of the BPLP protein orof its matured products.

In said above process, a half-saturating concentration is theconcentration of the BPLP protein or a maturation product thereof whichresults in a reduction by half of the degradation of the NEP substrate.

Another object of the present invention comprises a process forscreening a compound that is an antagonist of the BPLP protein or amaturation product thereof, comprising the steps of:

a) preparing a cell culture or preparing an organ specimen or a tissuesample (cryosections or slices or membrane preparations or crudehomogenates) containing NEP binding sites for the BPLP protein or amaturation product thereof;

b) incubating the cell culture, organ specimen or tissue sample of stepa) at concentrations allowing measurement of NEP enzymatic activityunder initial velocity conditions in the presence of a submaximalconcentration of the BPLP protein or a maturation product thereof (orany peptide retaining the binding specificity or the physiologicalactivity of the BPLP protein or of its matured products) and a NEPsubstrate, in the presence of the candidate compound during a timesufficient for the endoproteolysis of the NEP substrate to take placeunder initial velocity conditions;

c) quantifying the activity of the NEP present in the biologicalmaterial of step a) by measuring the levels of NEP substrateendoproteolysis, respectively in the presence or in the absence of thecandidate compound and in the presence or in the absence of the BPLPprotein or a maturation product thereof, or the peptide retaining thebinding specificity or the physiological activity of the BPLP protein orof its matured products.

In a preferred embodiment of said above process, a submaximalconcentration is a concentration of peptide which results in a reductionby at least 50% and preferably by at least 75% of the degradation of thesubstrate.

The below examples and figures illustrate the invention without limitingits scope.

LEGENDS TO THE FIGURES

FIG. 1 shows representative cation-exchange HPLC profile of ³H-YQRFSR(SEQ ID NO: 4) marker added to 2.5 ml salivary methanol-acid extractcorresponding to 2.5 ml human saliva. The recovery of the majorradioactive peak was evaluated at 75-84% (dotted bars).

FIG. 2 shows representative cation-exchange HPLC profile of a salivarymethanol-acid extract obtained from 7 ml human saliva. Fractions wereanalyzed for their inhibitory potency of substance P endoproteolysis byhuman ecto-endopeptidase activity (LNCaP cell line).

FIG. 3 is a representative reverse phase HPLC profile of the majorHPLC-EC active 13-14 fractions (dotted bars). Fractions were analyzedfor their inhibitory potency of substance P endoproteolysis by humanecto-endopeptidase activity (LNCaP cell line).

FIG. 4 is a representative reverse phase HPLC profile of the majorHPLC-RP active fractions. Fractions were analyzed for their inhibitorypotency of substance P endoproteolysis by human ecto-endopeptidaseactivity (black bars) and their absorbance at 274 nm (black line).

FIG. 5 shows the effect of BPLP-QRFSR peptide on the breakdown ofsubstance P by human ecto-endopeptidase activity (LNCaP cell line), theeffective concentration of QRFSR peptide (SEQ ID NO: 3) ranged from 1 to25 μM and being half-maximal at 11 μM.

FIG. 6 shows the effect of YQRFSR (SEQ ID NO: 4) derivative ofhBPLP-QRFSR peptide (SEQ ID NO: 3) on the breakdown of substance P byhuman ecto-endopeptidase activity (LNCaP cell line), the effectiveconcentration of YQRFSR peptide (SEQ ID NO: 4) ranged from 5 to 50 μMand being half-maximal at 30 μM.

FIG. 7 shows the effect of YQRFSR (SEQ ID NO: 4) derivative ofhBPLP-QRFSR peptide (SEQ ID NO: 3) on the breakdown of substance P byrat NEP ecto-endopeptidase activity (renal tissue), the effectiveconcentration of YQRFSR peptide (SEQ ID NO: 4) ranged from 5 to 75 μMand being half-maximal at 38 μM.

FIG. 8 is a RP-HPLC chromatographic analysis of the YQRFSR peptide (SEQID NO: 4). The YQRFSR peptide (SEQ ID NO: 4) (175 μM) was notmetabolized by human cell surface endopeptidases, in vitro, whilst itinhibited by 70% the substance P endoproteolysis mediated by human NEPectoendopeptidase. The RP-HPLC chromatographic characteristics revealedthat:

1/ the YQRFSR peptide (SEQ ID NO: 4) is not metabolized by human cellmembranes containing NEP; 93% was recovered as intact peptide against94% in absence of metabolizing membranes;

2/ in the same experimental conditions the YQRFSR peptide (SEQ ID NO: 4)inhibits by 70% the endoproteolysis of substance P by these human cellmembranes.

FIG. 9 shows the inhibitory effect of QRFSR-peptide (SEQ ID NO: 3) onthe breakdown of substance P by recombinant human NEP.Concentration-dependent inhibitory effect of QRFSR-Peptide (SEQ ID NO:3) on soluble recombinant human NEP activity and no effect ofQRFSR-peptide (SEQ ID NO: 3) on the endoproteolysis of substance P bysoluble recombinant hDPPIV activity.

FIG. 10 shows the inhibitory effect of QRFSR-peptide (SEQ ID NO: 3) onthe breakdown of APN synthetic substrate by cell surface human APN.Concentration-dependent inhibition by QRFSR-peptide (SEQ ID NO: 3) ofthe cleavage of Ala-pNA chromogenic substrate by cell surface HEK-hAPN.

FIG. 11 shows the inhibitory effect of QRFSR-peptide (SEQ ID NO: 3) onthe breakdown of NEP synthetic substrate by cell surface human NEP.Concentration-dependent inhibition by QRFSR-peptide (SEQ ID NO: 3) ofthe cleavage of Mca-BK2 fluorogenic substrate by cell surface HEK-hNEP.

FIG. 12 shows the in vivo effect of YQRFSR-peptide (SEQ ID NO: 4) on thetime spent by rat in paw licking of the formalin-injected hind paw;Mean±SEM.

FIG. 13 shows the in vivo effect of YQRFSR-peptide (SEQ ID NO: 4) on thenumber of pain spasms following hind paw formalin injection; Mean±SEM.

FIG. 14 shows the in vivo effect of YQRFSR-peptide (SEQ ID NO: 4) on theindex of pain spasms during the 60 minute post injection of formalin.The analgesia induced by QRFSR-derived peptide requires the activationof endogenous opioid receptors.

EXAMPLES

The study was designed to search natural metallo-ectopeptidases,especially NEP and/or APN inhibitor particularly in the human salivarysecretions. The strategy for the detection and isolation of this productwas based on the isolation of salivary low-molecular-mass components,which inhibit the endoproteolysis of NEP-sensitive substrate by humancells expressing the membrane-anchored human NEP. The inventors havedeveloped the models of functional detection (membranes preparations ofLNCaP and HEK human cells expressing NEP) and of molecular isolation(HPLC chromatography systems), for the identification by sequenceanalysis of the natural endogenous NEP ectopeptidase inhibitor(s) inhuman, i.e., the endogenous salivary functional homologue(s) of the ratsialorphin.

Example 1 Human Saliva Preparation

The protocol of clinical research established with the “centre derecherche Vaccinale et Biomedicale” of the Pasteur Institute, assessionnumber: 2045, received the agreement of the CCPPRB committee(PARIS-COCHIN) and samplings of the human saliva from 10 healthy malevolunteers, began in May 2003 and continued in October 2003. The salivawas collected into previously cooled “microsorp” tubes containingaprotinin (1000 KIU/ml) Pefabloc (0.4 mM) and HCl (0.1N) finalconcentration; this medium assuming to inhibit proteolysis activities.Thus saliva samples were stored at −80° C. until the methanol-extractionprocedure was performed.

Example 2 Materials and Experimental Models for NEP Inhibition

1-Sources of Human Ectopeptidases NEP and APN:

Several Human cell lines have been described as expressing NEP as wellas other members of the metalloecto-peptidase family; among them thereare an osteoblaste cell line, MG-63 (osteosarcoma), a trophoblaste cellline, BeWo (placental choriocarcinoma), an prostate epithelial cellline, LNCaP (adenocarcinoma) and an enterocyte cell line, Caco-2(colorectal adenocarcinoma). Culture conditions in defined medium usefulfor the cellular pharmacology analyses were first developed. Secondly,the inventors have confirmed by using Northern blot andimmunocytochemical analyses that the LNCaP and BeWo were the only celllines able to express NEP (ARNm and cell surface protein) in definedmedium culture conditions (i.e., RPMI containing insulin, transferin andselenium, GIBCO) and after induction by DHT (dihydrotestosterone) andforskolin, respectively. And finally, in the experimental model ofstatic incubations of membrane preparations originating from thesecells, the inventors have defined the parameters allowing to analyze thehuman NEP-mediated endoproteolysis of substance P in the conditions ofinitial velocity measurement, i.e. 100 pM/min/μg LNCaP cell membraneproteins (10-fold lower specific activity for BeWo). The LNCaP membraneactivity was inhibited in the presence of specific synthetic NEPinhibitor, such as thiorphan (62% for maximum inhibitory potency at 500nM). In contrast, bestatin (25 μM) and captopril (10 μM) which block theaminopeptidase (APN, APB.) and angiotensin-converting enzyme (ACE)activities, respectively, did not inhibit the substance P hydrolysis bycell surface ectopeptidases; thus indicating that in the experimentalconditions, the extra cellular breakdown of substance P was mainlycaused by the NEP endopeptidase activity located at the surface of thesecells.

In addition, in vitro model using the membrane preparations oftransfected HEK cells with human NEP cDNA or human APN cDNA (HEK cellsdo not express these metalloectopeptidases) and soluble recombinanthuman NEP or soluble recombinant human DPP IV (DipeptidylaminopeptidaseIV) (without the N-terminal cytosol and transmembrane segment) have alsobeen developed.

2-Substrates and Inhibitors:

In vitro, membrane amino- and endo-ectopeptidase activities of humancell membranes are assayed in vitro by measuring the breakdown of thefollowing synthetic and natural substrates:

a/Synthetic specific fluorogenic or chromogenic substrates:

(SEQ ID NO: 12) Mca-R-P-P-G-F-S-A-F-K (Dnp)-OH    and/or Suc-A-A-F-Amc(SEQ ID NO: 13)  (NEP) (R&D systems and Bachem)Ac-A-Amc or Ala-pNA(APN) (Bachem)

b/Physiological substrates:

(SEQ ID NO: 14) Modified tritiated substance P[(3,4³H)Pro²-Sar⁹-Met(O₂)¹¹]-Substance P(DuPont-NEN) and Native Substance   P: R-P-K-P-Q-Q-F-F-G-L-M(NEP-DPPIV-ACE) (Peninsula-Biovalley) (SEQ ID NO: 15)Native Met-enkephalin: Y-G-G-F-M (NEP-APN) (Peninsula-Biovalley)Measuring the hydrolysis of these substrates by cell-membrane peptidasesin the presence and absence of different available selective syntheticpeptidase inhibitors assessed the specificity of the peptidase assay:

-   -   Thiorphan, Phosphoramidon (NEP) (Sigma and Roche)    -   Bestatin, Amastatin (APN) (Calbiochem)    -   DPPIV inhibitor II (DPPIV) (Calbiochem)    -   Captopril (ACE) (Sigma)

3-Measurement of Peptidase Activities

The ectopeptidase activities were measured according to the protocoldeveloped and established for the functional characterization of the ratsialorphin (Rougeot et al., 2003). Briefly, for membrane preparations,the cells were homogenised at 4° C. in 10 volumes (vol./wt.) of 50 mMTris/HCl buffered at pH 7.1. A first centrifugation at 1000×g and 5° C.for 5 min allows to remove the cellular debris and the nuclei in thepellet. A second centrifugation at 100 000×g and 5° C. for 30 minconcentrates the membrane fraction in the pellet, which will besuperficially washed three times in cold Tris/HCl buffer, resuspended infresh buffer, aliquoted and stored at −80° C. while waiting to be usedas enzyme source.

Proteins determination was carried out using the Bio-Rad DC proteinassay with Bovine Serum Albumin (BSA) as the standard.

Hydrolysis of substrates was measured by monitoring the metabolism ratein conditions of initial velocity measurement in the presence andabsence of specific inhibitors. These were added to the preincubationmedium. The standard reaction mixture consisted of cell membranes in afinal volume of 200 μl Tris-HCl 50 mM pH 6.5-7.2. The substrate wasadded after preincubation for 10 min and the digestion carried out for20 min at 25° C. in a constantly shaken water bath. The reaction wasterminated by cooling to 4° C. and adding HCl (0.3N finalconcentration). The reaction tubes were then centrifuged (4700×g for 15min at 4° C.) and the remaining intact substrate and its metabolitesmeasured.

In the case of the use of natural substrates, substance P orMet-enkephalin, the products of the reaction are isolated and quantifiedaccording to their differential hydrophobic characteristics:

-   -   C-18 Sep-Pak cartridges (Waters) were used to analyse the        hydrolysis of radiolabeled substance P. The ³H metabolites were        isolated by elution with H2O-0.1% TFA and then with 25%        methanol-0.1% TFA (4 ml each). The intact tritiated substrate        was eluted with 75-100% methanol-0.1% TFA (4 ml).    -   RP-HPLC coupled to a spectrophotometer was used to analyse the        hydrolysis of Met-enkephalin, (C-18 LUNA column, AIT). Elution        with a 30-min linear gradient from 0.1% TFA in water to 0.1% TFA        in 100% acetonitrile, at 1 ml/min, separated the two        Met-enkephalin metabolites (YGG: 5.8±0.2; FM: 12.8±0.1 min        retention time) and the intact substrate (YGGFM: 18.8±0.2 min).        Their identities and relative quantities (peak height) were        checked by monitoring the column outflow at 264 nm (L3000,        Merck).    -   The disappearance of the initial Met-enkephalin substrate was        also quantified by radioimmunoassay (RIA). The assay used        anti-Met enkephalin antiserum (Gros et al., 1978) and        ¹²⁵I-Met-enkephalin (80 TBq/mmol, NEN); it detected nanomolar        concentrations of Met-enkephalin in the presence of micromolar        concentrations of Tyr-Gly-Gly and Phe-Met metabolites. The        radioactivity of each fraction was determined by liquid        scintillation spectrometry.

In the case of the use of synthetic substrates, the kinetics ofappearance of the fluorescent signal (intensity and polarization) wasdirectly analyzed by using a multi-well spectrofluorimeter; theintensity of the signal is directly proportional to the quantity ofmetabolites formed during the reaction.

Example 3 Human Saliva Purification and Chromatography

The protocol of extraction and purification of the human salivarycomponents mimicked the one that was developed and established for themolecular characterization of the sialorphin from rat saliva (Rougeot etal., 1994), and the extracts and chromatographic fractions were analyzedfor their capacity to inhibit the hydrolysis of the physiologicalsubstrate, substance P, by the human cell membranes containing NEP.

Extraction and purification of the human salivary compounds potentiallyregulators of enkephalinase activity. Briefly, following defrosted at+4° C., the saliva samples were treated according to the followingprocedure:

-   -   Methanol-acid extraction procedure: Extraction of low        molecular-mass components in methanol-acid at 4° C.; to 1 volume        of saliva was added 4 volumes of methanol containing 0,1%        trifluoroacetic acid (TFA) solution. This first step realizes        the elimination of proteins of high molecular weight (including        the degrading enzymes), which are inactivated and precipitated        in acid and methanol medium respectively and allows the        solubilization of the salivary constituents of small molecular        weight (≦10 Kda). The methanol mixture was quickly vortexed and        centrifuged for 15 min at +4° C. and 12OOO g; the methanol was        removed from the supernatant after lyophilization at −110° C.    -   HPLC cation-exchange chromatography (HPLC-EC): The        methanol-extracted saliva was solubilized in the solvent A,        i.e., ammonium acetate 10 mM pH 4.3, and injected into a        HEMA-IEC BIO-1000 carboxymethyl column (Alltech). Components        were eluted and isolated according to their cationic        characteristic, in a two-step linear gradient of 10-500 mM and        500-900 mM ammonium acetate pH 4.7, respectively and at a 1        ml/min flow rate. Fractions of 2 ml were collected and tested        after lyophilization for their inhibitory potency of the human        ectopeptidase activity (LNCaP).

Quality and recovery of extraction and successive chromatographies wereestimated using an internal standard (the tritiated peptide: 3H-YQRFSR)added to a representative salivary sample, as illustrated in FIG. 1; therecovery of the marker added to sample extracted corresponding to 2.5 mlof human saliva was evaluated at 75-84%. HPLC cation-exchangechromatography of methanol-extracted saliva (FIG. 2; representativeprofile of a salivary extract corresponding to 7 ml of human saliva)clearly revealed the presence of two major molecular salivarycomponents, which were eluted within the first-step ammonium acetategradient profile (10-500 mM) at retention times of 26-28 and 36-38 minrespectively and that inhibited by ≧90% the endoproteolysis of substanceP by human membrane-bound peptidases (The 2 active peaks visualized FIG.2 with the retention times of 6 and 48 min correspond to the exclusionand total volume of the column, respectively).

-   -   HPLC reverse-phase Chromatographies (RP-HPLC). The active        fractions of the previous HPLC-EC were solubilized in the        solvent A [0,1% TFA in H2O] and injected into a Synergi Max-RP        column (Phenomenex). Sample components were eluted (1 ml/min)        with a linear gradient of 1-99% solvent B [acetonitrile-TFA,        100-0.1, by vol.]. Fractions of 1 ml were collected and analyzed        after lyophilization for their inhibitory potency towards the        cell surface human ectopeptidase activity (LNCaP). The recovery        of the internal marker was evaluated at 61%. Fractionation by        RP-HPLC (FIG. 3), of the active molecular forms isolated from        fractions 13-14 (26-28 min-retention time) of the previous        HPLC-EC, showed the presence of two major molecular populations        inhibiting the human endopeptidase activity, and that were        eluted within the acetonitrile gradient profile at retention        times of 23-25 and 28-30 min, respectively.

These fractions underwent further purification procedure on a newsynergi Max-RP-HPLC column through elution with a linear gradient of1-99% solvent B [100% methanol-0.1% TFA]. Column eluates were collectedin microsorb tubes at 1-min intervals and the fractions were testedafter lyophilisation for their NEP inhibitory activity. As shown in FIG.4, two major molecular forms, which inhibited the endoproteolysis ofsubstance P by human ectopeptidases, were thus isolated with retentiontimes of 20-21 and 29-30 min respectively, and their amino acidsequences were determined.

-   -   Ciphemen ProteinChip and amino-acid sequence analyses.        N-terminal sequence analysis was performed by automated Edman        degradation using Applied Biosystems peptide sequanators        (plate-forme d'Analyse et de Microséquençage des Protéines,        Institut Pasteur). The molecular form eluting from the ultimate        RP-HPLC at 18 min-retention time (fraction 20) corresponded to        690 and 769.5 Da molecular mass and to the following sequence of        five amino acid residues: QRFSR (SEQ ID NO: 3). That one eluting        at 26 min-retention time (fraction 28) corresponded to two        molecular components of 622-666 Da and 6495 Da, respectively;        the amino-acid determination of the highest molecular mass        indicated that it corresponds to a salivary Basic Proline-Rich        Polypeptide sequence, the human PRP-E of 61 amino-acid sequence        (Isemura et al., 1982).

By analogy with the rat salivary sialorphin, these data provide directevidence for the existence of a human salivary sialorphin-like, a QRFSRpentapeptide (SEQ ID NO: 3) of structure and function closely related tothose of rat QHNPR pentapeptide (SEQ ID NO: 8) and which is secretedinto the human salivary secretions; they support that QRFSR (SEQ ID NO:3) is the mature product proteolytically processed from a precursorprotein in a fashion similar to the maturation pathway of SMR1 andpeptide-hormone precursors. Furthermore, as for the QHNPR rat peptide(SEQ ID NO: 8), the excreted QRFSR peptide (SEQ ID NO: 3) seems to beaccumulated in the human salivary secretions under different forms,among which the free forms including probably an acetate salt form andthe complex forms involving high hydrophobic interactions with salivaryPRP-E.

Example 4 Synthesis and Testing of QRFSR (SEQ ID NO: 3) Peptide

The QRFSR peptide (SEQ ID NO: 3) was synthesized and analyzed for itscapacity to inhibit the degradation of the physiological NEP substrate,the substance P, in vitro, in the experimental model of staticincubation of human LNCaP cell membranes. The peptide QRFSR (SEQ ID NO:3), inhibited the extra-cellular endoproteolysis of substance P mediatedby human NEP expressed at the surface of human prostate epithelialcells. The effective concentration for QRFSR ranged from 1 to 25 μM, andbeing half-maximal (IC50) at 11 μM (FIG. 5). Surprisingly, but inredundant way with regard to what was observed with rat sialorphintowards the human NEP, the inhibitory efficiency of the QRFSR humanpeptide (SEQ ID NO: 3) towards the rat renal NEP activity is at least10-fold lower than that obtained towards the human cell surface NEP(LNCaP). Strikingly, the derivative peptide YQRFSR (SEQ ID NO: 4), whichhas been synthesized for tritium labeling and immunogenic conjugationfor the development of antibody and immunoassay detection system,appeared to exhibit a relatively similar inhibitory efficacy towardsboth human and rat ecto-endopeptidase activities (FIGS. 6 and 7).

Table: inhibitory potency of natural and derivative human and ratpeptides towards both human and rat ectoendopeptidase activities:

Ectoendopeptidase from Human cells Rat tissues QHNPR (SEQ ID NO: 8)  4 to 40 μM 0.4 to 4 μM QHNP (SEQ ID NO: 9) undetermined ≧50 μMQRFSR (SEQ ID NO: 3) 2.5 to 25 μM ≧100 μM YQRFSR (SEQ ID NO: 4)  5 to 50 μM 5-75 μM QRGPR (SEQ ID NO: 10) ≧90 μM undetermined QRGPRGP (SEQ ID NO: 11) ≧90 μM undetermined

Besides, the QRGPR peptide (SEQ ID NO: 10) (20-90 μM) which could bepotentially maturated from hPB gene products, had no effect on substanceP endoproteolysis induced by LNCaP human cell membranes; this resultlets the inventors to propose that the nature of three central aminoacids of the natural NEP-inhibitor pentapeptide (common Q-Nterminal andR-Cterminal) is determining signature for the affinity and/orspecificity of their functional interaction with NEP ectoendopeptidase.Furthermore, in spite of the strong primary amino-acid sequence analogybetween the rat and human NEP (≠85%), the inventors observed a relativespecificity in the functional interaction of both naturalinhibitor-pentapeptides, respectively the rat QHNPR (SEQ ID NO: 8) andhuman QRFSR (SEQ ID NO: 3). All these results provide evidence for theexistence of a conformational specificity in the secondary and tertiaryof both ectoenzymes; the crystal structure determination of the binarycomplex formed with the sialorphin or its derivatives and the human NEPshould allow to gain insight into the binding mode of these naturalcompetitive inhibitors.

The inventors used the tritiated 3H-YQRFSR peptide (SEQ ID NO: 4) toestablish the pharmacokinetic and pharmacodynamic parameters, of thishuman functional peptidomimetic of rat sialorphin in vivo in adult malerat (biodistribution-bioavailability-clearance) as well as to define itsmetabolism mechanism and turnover in vivo and in vitro, (FIG. 8). TheRP-HPLC chromatographic characteristics revealed that:

-   -   the YQRFSR peptide (SEQ ID NO: 4) is not metabolized by human        cell membranes containing NEP, indeed 93% was recovered as        intact peptide against 94% in absence of metabolizing membranes,    -   in the same experimental conditions, the YQRFSR peptide (SEQ ID        NO: 4) inhibits by 70% the endoproteolysis of substance P by        these human cell membranes.

Therefore, YQRFSR (SEQ ID NO: 4) is useful for investigating theanalgesic activity of the BPLP maturation products in behavioral ratmodels of acute pain, e.g., the Pin pain test and Formalin test, whichhave been studied for the functional characterization of the sialorphinin vivo (Rougeot et al., 2003).

Example 5 Further Characterization of QRFSR Peptides In Vitro

The inhibitory specificity of the QRFSR-peptide (SEQ ID NO: 3) wasassessed by measuring the endoproteolysis of substance P(SP) in an invitro enzyme-assay using purified soluble human NEP and human DPPIV(without the N-terminal cytosol and transmembrane segment). Using theselective recombinant hNEP assay, the molecular interaction of humanQRFSR-peptide (SEQ ID NO: 3) with hNEP was established, providing directevidence that the peptide inhibited hNEP activity: as shown on FIG. 9,QRFSR-peptide (SEQ ID NO: 3) prevented the NEP mediated-endoproteolysisof SP by 90%; its inhibitory potency was strictly concentrationdependent (r²=0.99, n=18), ranged from 5 to 50 μM and was half-maximalat 29±1 μM. In contrast, the breakdown of SP by recombinant hDPPIV wasnot prevented by 25 or 50 μM QRFSR-peptide (SEQ ID NO: 3), indicatingthat the inhibitory potency of the QRFSR-peptide on the SP-catabolizingcell surface ectoenzymes in vitro, is simply due to its specificinteraction with NEP-ectopeptidase. Furthermore, from studies monitoringthe in vivo metabolism of SP, it appears likely that the QRFSR-peptide(SEQ ID NO: 3), like rat QHNPR-sialorphin (SEQ ID NO: 8), does notentirely protect endogenous SP from cleavage by the spinalSP-inactivating ectopeptidases, and therefore would not potentiateSP-mediated nociception in vivo.

The enkephalins are inactivated in vivo with remarkable efficiency(within a few seconds) by both ectopeptidases, NEP and APN. Owing to thecomplementary role of NEP and APN in enkephalin inactivation, only mixedNEP-APN synthetic inhibitors induce antinociceptive responses in variouspain models.

Thus, the inhibitory specificity of QRFSR-peptide (SEQ ID NO: 3) wasassessed in an enzyme-assay using membrane preparations of recombinantHEK human cells expressing selectively either human membrane-anchoredNEP or APN. These transfected-cell models were developed in thelaboratory. Membrane amino- and endo-ectopeptidase activities of humancell membranes were assayed in vitro by measuring the breakdown ofartificial specific fluorogenic substrates, the NEP substrate used was:Mca-R-P-P-G-F-S-A-F-K-(Dnp)-OH (SEQ ID NO: 12) (Mca-BK2) and the APNsubstrate was: Ala-pNA. Using the selective membrane-anchored hNEPassay, the inventors found that the inhibition by the QRFSR-peptide (SEQID NO: 3) of Mca-BK2 endoproteolysis by NEP is concentration dependent(r²=0.88, n=29 determination points) and the effective doses ranged from5 to 50 μM. Using the selective membrane-anchored hAPN assay, theinventors have demonstrated that QRFSR-peptide (SEQ ID NO: 3) inhibitsthe Ala-pNA cleavage by hAPN at 10 to 90 μM effective doses (r2=0.93,n=22 determination points) (see FIGS. 10 and 11).

TABLE 1 Summary of QRFSR (SEQ ID NO: 3) inhibitory effects (IC₅₀) on NEPand APN ectoenzyme activities, in vitro and ex vivo: Enzymes IC₅₀ valuessources Substrate QRFSR-peptide HEK-hNEP Substance P (60 nM) 14 μMMcaBK2 (5 μM) 33 ± 6 μM LNCaP Substance P 11 ± 3 μM McaBK2 25 ± 1 μMhNEP soluble Substance P 29 ± 1 μM HEK-hAPN Ala-pNA (100 μM) 65 ± 9 μM

These results indicate that the human QRFSR-pentapeptide (SEQ ID NO: 3)is an efficient dual inhibitor of NEP and APN ectopeptidase activities,in vitro. Furthermore, owing to the complementary role of NEP and APN inenkephalin inactivation and by analogy with rat sialorphin which exertsa powerful analgesic activity, the combined biological and genomicinformation accrued led the inventors to propose that the QRFSR-peptide(SEQ ID NO: 3), by inhibiting enkephalin-inactivating NEP-APNectopeptidases, potentiates enkephalin-dependent antinociceptivemechanisms, in vivo.

Example 6 Functional Characterization of QRFSR (SEQ ID NO: 3) Peptide InVivo

In spite of the strong primary amino-acid sequence analogy between therat and human NEP (≠85%), the inventors observed a relativespecies-selectivity in the inhibitory potency of bothinhibitor-pentapeptides, respectively the rat QHNPR (SEQ ID NO: 8) andhuman QRFSR (SEQ ID NO: 3). Strikingly, the derivative peptide YQRFSR(SEQ ID NO: 4), which was synthesized for tritium labeling, appeared toexhibit a relatively similar inhibitory efficacy towards both human andrat ectoendopeptidases (range of effective concentrations between 5 and50 μM). Thus, the antinociceptive potency of the QRFSR-derived peptide(SEQ ID NO: 3) was investigated in the behavioral rat model of acutepain, i.e., the formalin test, which was used for the in vivocharacterization of rat sialorphin action (Rougeot et al., 2003).Systemic administration of 0.5 and 1 mg/kg YQRFSR-peptide (SEQ ID NO: 4)inhibited the early phase (first 20 min after formalin injection) of pawlicking of the formalin-injected hind paw. For instance, itsignificantly reduced the time spent by treated rats in paw licking from144±17 s, n=8 (vehicle) to 97±14 s, n=8 (0.5 mg/kg) (p=0.05) and to84±13 s, n=8 (1 mg/kg) (p=0.02 by Dunnett t-Test). Surprisingly, incontrast to rat sialorphin-treated rats, the YQRFSR peptide-treated ratsspent significantly less time in paw licking during the late phase (40to 60 min after formalin injection) of the formalin test(vehicle-treated rats: 63±13 s vs. 1 mg/kg treated-rats: 9±3 s,p=0.001). Although less potent than rat sialorphin, in term of effectivedoses (100-200 μg/kg, iv), the QRFSR-derived peptide (SEQ ID NO: 3)seems to be as efficient in its pain-suppressive potency (1 mg/kg, iv),as the synthetic mixed NEP-APN inhibitor RB101 (2.5-5 mg/kg, iv) in theformalin-induced pain model.

These data (as presented on FIGS. 12, 13 and 14) clearly indicate thatthe YQRFSR-peptide (SEQ ID NO: 4) inhibits nociception induced by acuteand long-acting chemical stimuli.

Its analgesic potency is almost as efficient as 3 mg/kg morphine dose.

Furthermore, the analgesia induced by the QRFSR-derived peptide (SEQ IDNO: 3) in the chemical-evoked pain behaviour is totally reversed in thepresence of an opioid receptor antagonist, the nalaxone, which isconsistent with an involvement of the endogenous opioidergic pathways inits analgesic effect.

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1. An in vitro method for prognosis, diagnosis or determination of theevolution of a condition involving an altered production of BasicProline-rich Lacrimal Protein (BPLP) or of any of its maturationproducts, which method comprises detecting, or quantifying in abiological sample of a test subject, a BPLP protein or a maturationproduct thereof, and comparing the production of BPLP protein ormaturation product with the production of the same in a biologicalsample of a control subject.
 2. The method according to claim 1, whereinsaid BPLP consists of the sequence SEQ ID NO:
 2. 3. The method accordingto claim 1, wherein the maturation product is a peptide that is obtainedthrough cleavage of the BPLP protein precursor by prohormones convertingenzymes or natural maturases.
 4. The method according to claim 3,wherein cleavage of the BPLP protein precursor is obtained by furin, aPC concertase or PACE4.
 5. The method according to claim 3, wherein thematuration product comprises the sequence QRFSR (SEQ ID NO: 3) andwherein said sequence is the C-terminal part of the maturation product.6. The method according to claim 5, wherein said peptide consists ofsequence QRFSR (SEQ ID NO: 3).
 7. A method for a detection of theproduction of Basic Proline-rich Lacrimal Protein (BPLP) or of any ofits maturation products, which method is performed by contacting abiological sample with an antibody that specifically recognizes apeptide that is a maturation product of the BPLP.
 8. The methodaccording to claim 7, wherein said peptide is a maturation productobtained through cleavage of the BPLP protein precursor by prohormonesconverting enzymes or natural maturases.
 9. The method according toclaim 8, wherein cleavage of the BPLP protein precursor is obtained byfurin, a PC concertase or PACE4.
 10. The method according to claim 7,wherein said peptide comprises the sequence QRFSR (SEQ ID NO: 3) andwherein said sequence is the C-terminal part of the maturation product.11. The method according to claim 10, wherein said peptide consists ofsequence QRFSR (SEQ ID NO: 3).
 12. A method for a detection of theproduction of Basic Proline-rich Lacrimal Protein (BPLP) or of any ofits maturation products, which method is performed by contacting abiological sample with an antibody that specifically recognizes the BPLPprotein.
 13. The method according to claim 12, wherein said BPLPconsists of the sequence SEQ ID NO: 2.